Intra-nasal administration of the B Subunit of the E. coli heat labile enterotoxin (EtxB) has been shown to protect against the development of collagen-induced arthritis (CIA) in DBA/1 mice and diabetes in NOD mice. Protection from autoimmunity could be transferred with splenic CD4+ T cells and was not associated with a shift towards TH2-associated cytokines or antibody isotypes, implying that EtxB induces or modulates the activity of regulatory CD4+ T cells (Treg). In order to elucidate the mechanisms involved, a number of parameters have been studied in vivo following EtxB administration. Immunohistochemical analysis of the distribution of EtxB showed that it can be found in local sites (nasalassociated lymphoid tissue (NALT) and the cervical lymph nodes (CLN)) as well as distant sites (spleen and inguinal lymph nodes). Direct fluorescent labelling of EtxB showed that it is bound to a variety of cell types in the CLN and spleen and to mononuclear cells in the blood. EtxB up-regulated expression of the antiinflammatory cytokines IL-10 and TGF-131 in the NALT, CLN and spleen without a concomitant increase in IFN-y expression and increased proportion of CD4+Foxp3+ T cells, primarily in the CD2S- fraction, in the CLN and spleen. Furthermore, EtxB was also capable of inducing an increase in the proportion of CD4+Foxp3+ T cells specific for a co-administered antigen, even when the antigen is administered in an inflammatory context. Therefore, following intra-nasal administration, EtxB reaches local and distant lymphoid organs, where it induces an anti-inflammatory cytokine milieu that is associated with an increase the proportion of CD4+Foxp3+ T cells. As Foxp3 expression confers a regulatory phenotype, this increase is likely to underlie the ability of EtxB treatment to protect from CIA and diabetes development.